Insulin signaling in skeletal muscle during inflammation and/or immobilisation

Background The decline in the downstream signal transduction pathway of anabolic hormone, insulin, could play a key role in the muscle atrophy and insulin resistance observed in patients with intensive care unit acquired weakness (ICUAW). This study investigated the impact of immobilisation via surgical knee and ankle fixation and inflammation via Corynebacterium parvum injection, alone and in combination, as risk factors for altering insulin transduction and, therefore, their role in ICUAW. Results Muscle weight was significantly decreased due to immobilisation [estimated effect size (95% CI) − 0.10 g (− 0.12 to − 0.08); p < 0.001] or inflammation [estimated effect size (95% CI) − 0.11 g (− 0.13 to − 0.09); p < 0.001] with an additive effect of both combined (p = 0.024). pAkt was only detectable after insulin stimulation [estimated effect size (95% CI) 85.1-fold (76.2 to 94.0); p < 0.001] irrespective of the group and phosphorylation was not impaired by the different perturbations. Nevertheless, the phosphorylation of GSK3 observed in the control group after insulin stimulation was decreased in the immobilisation [estimated effect size (95% CI) − 40.2 (− 45.6 to − 34.8)] and inflammation [estimated effect size (95% CI) − 55.0 (− 60.4 to − 49.5)] groups. The expression of phosphorylated GS (pGS) was decreased after insulin stimulation in the control group and significantly increased in the immobilisation [estimated effect size (95% CI) 70.6-fold (58.8 to 82.4)] and inflammation [estimated effect size (95% CI) 96.7 (85.0 to 108.5)] groups. Conclusions Both immobilisation and inflammation significantly induce insulin resistance, i.e., impair the insulin signaling pathway downstream of Akt causing insufficient GSK phosphorylation and, therefore, its activation which caused increased glycogen synthase phosphorylation, which could contribute to muscle atrophy of immobilisation and inflammation. Supplementary Information The online version contains supplementary material available at 10.1186/s40635-023-00503-9.


Table of Contents
Detailed Methods 3 Table A1. Antibodies for Western Blot 7 Additional Results: Body weight per muscle weight 8 Figure A1. Body weight per Muscle weight in gram per gram 8 References 9 Detailed Methods

Sample size
Sample size calculation was based on (p)Akt concentrations. Sugita et al. [1] were able to show a reduction within a burn model to 53% and a standard error of the mean of 7% and 6%, resulting in standard deviations of 22.1% and 15.8%, respectively. We set a 25% reduction of the p-Akt/PKB activation as relevant difference to show insulin resistance. This led to a critical difference = 25% & (7% * *10) ! + (5% * *10) We set an alpha level of 0.003125 after correction for multiple testing (0.05/16 = 0.003125) with 16 different groups (8 different interventional groups and 2 legs). A beta value of 0.2 within a one-sided t-test leads to necessary number of legs of 20, i.e., 10 animals per treatment group. According to previous studies using this model we expected a drop-out rates of 15% due to the study procedures (e.g., surgery and anaesthesia), 10% due to insufficient immobilisation and 35% due to severity of inflammation and animal protection in the respective groups (e.g., 35% are only calculated in inflammation groups). We therefore arrive at a sample size of 132 animals of which 52 are reserves animals.

General housing
Rats were acclimatized to standard conditions of the animal laboratory for at least seven days. Animal housing facility was subject to a person-limited access control, air conditioning and a light-dark rhythm of 12 hours each. Animals were kept in a pathogen-free environment in macrolon cages type III and IV. Food and drinking water were available ad libitum. Body weight of animals was recorded, and their clinical condition was evaluated daily. Germany) was used. Animals in sham inflammation groups received 0.5 ml saline instead of Corynebacterium parvum. Otherwise, the procedure was identical.

Insulin injection
On day 12, animals were put under general anaesthesia after at least six hours of fasting (water unrestricted). Anaesthesia, analgesia, intubation, ventilation and monitoring were equivalent to procedures for immobilization. Fasting before insulin stimulation was important to generate fasting blood glucose levels for the experiment.
Then, the abdominal cavity was opened under deep isoflurane anaesthesia.
Subsequently, insulin groups received 0.65 IU/kg insulin injected via the portal vein.
Five minutes later, tibialis muscles of both legs were harvested, weighed, immediately frozen in liquid nitrogen and stored at -80°C for western blot analysis. Animals were then euthanized under deep general anaesthesia. Animals of sham insulin groups received saline instead of insulin. Otherwise, the procedure was identical.

Western Blot
Therefore, tibialis muscle was homogenized in RIPA lysis buffer containing protease inhibitor, using a tissue lyser (TissueRuptor II, Qiagen, Germany  The main factors immobilisation, inflammation and side (p < 0.001) had a significant effect. Furthermore, the interaction of side x immobilisation had a significant effect (p < 0.001).

Fig. A1 Body weight per Muscle weight in gram per gram
In all 4 groups a significant difference between the operated and contralateral leg was present (p < 0.001 for all four groups The number of identical symbols at the top of the error bars indicate the significance level: one < 0.05; two < 0.01 and three < 0.001. ✱ showed significant difference between the operated and contralateral leg within the same group # showed significant difference to the control group for the respective leg + showed significant difference to the immobilisation group for the respective leg § showed significant difference to the inflammation group for the respective leg